Ankara Üniv Vet Fak Derg, 63, 401-406, 2016

Pathomorphological evaluation of toxic effect of cypermethrin and

cyphenothrin in common carp*

Gözde YÜCEL, İbrahim Ayhan ÖZKUL

Ankara University, Faculty of Veterinary Medicine, Department of Pathology, Ankara, Turkey.

Summary: In this study, juvenile common carp (Cyprinus carpio) were subjected to sublethal concentrations of the synthetic

pyrethroids cypermethrin (2 µg/L) and cyphenothrin (5 µg/L) for 75 days. The toxic effects of these substances were analyzed

pathomorphologically by conducting necropsies on 7 fish on days 15, 30, 45, 60, and 75. In addition, histopathological examination

of the gonads was performed by exposing to 17β-estradiol (E2) (25 µg/L) as a positive control group to assess the endocrine

disrupting effects of cypermethrin and cyphenothrin. Hyperemia, telangiectasia, epithelial lifting, and branchitis were observed in the

gills of the fish from both treatment groups, and hemorrhage in the kidneys and hyperemia in the brain were also detected. Only 1

fish from each of the cypermethrin and E2 groups was observed to have degeneration of the testicles and edema of the ovary. One

fish from the cyphenothrin group had degeneration of the testicles and another had oophoritis of the ovary. Based on the hyperemia

and telangiectasia observed in the gills, it was concluded that both cypermethrin and cyphenothrin may lead to shortness of breath in

these fish. However, only minor lesions were detected in the gonads; thus, there was no evidence that these synthetic pyrethroids

affect the endocrine system.

Keywords: Cypermethrin, cyphenothrin, Cyprinus carpio (common carp), 17β-estradiol (E2), histopathology.

Cypermethrin ve cyphenothrinin sazan balıklarına toksik etkisinin patomorfolojik yönden

değerlendirilmesi

Özet: Bu çalışmada, sentetik piretroitlerden olan cypermethrin ve cyphenothrinin belirlenen sublethal dozları (2 µg/L ve 5

µg/L) juvenil sazan (Cyprinus carpio) balıklarına 75 gün süreyle uygulandı. Her gruptan yedişer balığın nekropsileri 15, 30, 45, 60

ve 75. günlerde yapılarak toksik etkileri patomorfolojik yönden değerlendirildi. Cypermethrin ve cyphenothrinin endokrin sisteme

olan etkilerini değerlendirmek amacıyla pozitif kontrol olarak 17β-estradiol (E2) (25 µg/L) verilerek gonadlar da histopatolojik

yönden incelendi. Cypermethrin ve cyphenothrin grubuna ait balıkların solungaçlarında hiperemi, telangiektazi, epitelde ayrılma ve

branşitis ile karşılaşıldı. Böbreklerde kanama ve beyinde hiperemi dikkati çekti. Gonadlarda ise cypermethrin ve E2 grubuna ait

sadece birer balıkta testiste dejenerasyon ve birer balıkta ovaryumda ödem ile karşılaşıldı. Cyphenothrin grubuna ait bir balıkta

ovaryumda ooforitis, bir balıkta ise testiste dejenerasyon görüldü. Solungaçlarda oluşan hiperemi ve telangiektazi bulguları birlikte

değerlendirildiğinde; cypermethrin ve cyphenothrinin balıklarda solunum yetmezliğine yol açabilecekleri sonucuna varıldı. Bu

çalışmada, gonadlarda az sayıda lezyon görülmesi nedeniyle sentetik piretroitlerin endokrin sisteme olan etkileri saptanmadı.

Anahtar sözcükler: Cypermethrin, cyphenothrin, Cyprinus carpio (sazan), 17β-estradiol (E2), histopatoloji.

Introduction

Pesticides are toxic substances that are used to kill

vermin or “pests.” These formulations are designed to

prevent, control, or diminish the destructive effects of

these harmful organisms. Where pesticides are used

accidentally or excessively, they can leave residues in the

environment. When used continuously, they accumulate

exponentially in the environment, plants, and other living

organisms (2, 5, 7, 10).

Pyrethroids, synthetic analogs of the natural

pyrethrins extracted from Chrysanthemum cinerariaefolium,

are used in pesticides. Cypermethrin [CAS No. 52315-

07-8, cyano- (3-phenoxyphenyl) methyl-3- (2,2-

dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate]

is a synthetic pyrethroid that is commonly used as a

pesticide (particularly against insects) to improve plant

health and is also used as an ectoparasitic medication for

animals (16). Cyphenothrin [CAS No. 39515-40-7, cyano

(3-phenoxyphenyl) methyl 2,2-dimethyl-3- (2-

methylprop-1-en-1-yl) cyclopropanecarboxylate] is

another synthetic pyrethroid that is commonly used

against flies, mosquitoes, and cockroaches (15).

Synthetic pyrethroids are considered safer than

organophosphates, carbamates, and organochlorine

* This study was prepared from PhD thesis of the first author.

Gözde Yücel - İbrahim Ayhan Özkul 402

compounds due to their lower persistence in the

environment. However, pyrethroids have also been

reported as being extremely toxic to aquatic organisms

(2, 10).

Freshwater and marine animals are commonly used

to analyze the ecotoxicology of chemical substances that

are suspected to leave residues. Fish are among the

creatures that are used for such analyses (9). In fish, 17β-

estradiol (E2), one of the natural estrogens that is

secreted by the ovaries, stimulates the liver to synthesize

vitellogenin, a protein that promotes the ripening of eggs

(6, 8, 12, 18). It is believed that chemical substances that

are expected to have estrogenic impact stimulate the

synthesis of vitellogenin in the liver, allowing its

degenerative effects to be identified in the gonads (12,

18).

In this study, we investigated the toxic effects of

synthetic pyrethroids on common carp (Cyprinus carpio)

by exposing fish to sublethal concentrations of

cypermethrin (2 µg/L) and cyphenothrin (5 µg/L) for 75

days. The tissues and organs of the fish were analyzed

pathomorphologically by conducting necropsies on 7 fish

on days 15, 30, 45, 60, and 75, and the gonads were

examined histopathologically by exposing to 17β-

estradiol (E2) as a positive control group to determine the

effects of these chemicals on the endocrine system.

Materials and Methods

Common carp (n = 140) were obtained from the

Public Waterworks Administration Amasya Water

Resources Research Institute Hatchery Station. The fish

had an average length of 15.51 cm and an average weight

of 69.63 g. The experiment was performed in the Ankara

University Faculty of Veterinary Medicine Department

of Pathology Experimental Unit (Ethic No: 2010-60-

310).

The carp were placed in 20 experimental aquaria at

a density of 7 fish/aquarium for 15 days before the start

of the experiment to allow them to acclimatize. Each

125-L aquarium was filled with 100 L of water, which

was aerated constantly.

Technical grade cyphenothrin (93.1%) and

cypermethrin (93%) were obtained from the Insecticide

Testing Laboratory of Hacettepe University, Ankara, and

technical grade 17β-estradiol (E2) (98%) was obtained

commercially. Sublethal concentrations of cypermethrin,

cyphenothrin and concentration of E2 were determined to

be 2 µg/L, 5 µg/L, and 25 µg/L, respectively, according

to pre-test results and the findings of previous studies.

All chemicals were stored at 4°C and brought to room

temperature before introduction to the experimental

setup. Doses of the chemicals were applied at 2-day

intervals to maintain the pesticide and E2 concentrations.

Seven fish from each experimental group were

euthanized on days 15, 30, 45, 60, and 75, followed by

necropsies. Tissue samples were fixed in 10% buffered

neutral formalin, processed routinely, and embedded in

paraffin blocks. These blocks were then cut into 4-6 μm

sections, which were stained with hematoxylin and eosin

(HE) and examined under a light microscope.

Results

Behavioral changes: Fish belonging to the control,

cypermethrin, and E2 groups all showed normal behavior

throughout the test period. The only unusual behavior

was exhibited by a single fish from the cyphenothrin

group, which was observed to swirl while swimming on

the first day of treatment.

Macroscopic findings: No significant macroscopic

findings were identified in any of the groups.

Microscopic findings: Fish from the control group

did not exhibit any histopathological changes in any of

the tissues examined (Figures 1a, 2a, 3a, 4a, 4c).

Similarly, fish exposed to cypermethrin and cyphenothrin

did not exhibit any significant histopathological changes

in the tissues of the skin, spleen, stomach, intestines,

heart, and skeletal muscles.

Cypermethrin: In the gills, hyperemia was observed

in the central venous sinus (Figure 1b), artery, and

capillary lumen. Telangiectasia was evident in the

capillaries of the secondary lamellae (Figure 1c);

epithelial lifting was also observed in the secondary

lamellae (Figure 1d). Hemorrhage was seen. Branchitis

was indicated by the presence of lymphocytes and

macrophages in the primary and secondary lamellae of

the gills (Figure 1b). Passive hyperemia and hydropic

degeneration were seen in the livers of a few fish, and

hemorrhage was observed in the kidneys of some. In the

brain, hyperemia and focal gliosis (Figure 2b) were

observed. Table 1 presents the number of fish in the

cypermethrin group showing microscopic changes on

each day.

In the gonads, a fish appeared to experience

interstitial edema in the ovary on day 60, whereas

degeneration of the seminiferous tubules of testicle was

observed in one other fish on day 15 and in 2 fish on day

45.

Cyphenothrin: In the gills, hyperemia, telangiectasia,

epithelial lifting, and branchitis were observed. Passive

hyperemia and hydropic degeneration were also seen in

the liver (Figure 3b), and some hemorrhage was observed

in the kidneys of some fish. In the brain, hyperemia and

focal gliosis were observed. Table 2 presents the number

of fish in the cyphenothrin group showing microscopic

changes on each day.

In the gonads, 1 female fish had oophoritis with

infiltration of lymphocytes and macrophages in the

interstitial area of the ovaries on day 30 (Figure 4b),

whereas the lumens of the seminiferous tubules were

dilated in 1 male fish on day 15 (Figure 4d).

Ankara Üniv Vet Fak Derg, 63, 2016 403

Figure 1.

a. Gill, control group, bar = 100 µm, HE.

b. Branchitis composed of mononuclear cells (arrow head), hyperemia in central venous sinus (white arrow), cypermethrin group,

45th day, bar = 100 µm, HE.

c. Telangiectasia in the capillaries of the secondary lamellae (star), cypermethrin group, 15th day, bar = 50 µm, HE.

d. Epithelial lifting in the secondary lamellae (black arrow), cypermethrin group, 15th day, bar = 50 µm, HE.

Şekil 1.

a. Solungaç, kontrol grubu, bar = 100 µm, HE.

b. Mononüklear hücrelerin oluşturduğu branşitis (ok başı), sentral venöz sinüsteki hiperemi (beyaz ok), cypermethrin grubu, 45. gün,

bar = 100 µm, HE.

c. Sekonder lamella kapillar lümenlerinde telangiektazi (yıldız), cypermethrin grubu, 15. gün, bar = 50 µm, HE.

d. Sekonder lamella epitellerinde ayrılma (siyah ok), cypermethrin grubu, 15. gün, bar = 50 µm, HE.

Figure 2.

a. Brain, control group, bar = 50 µm, HE.

b. Focal gliosis (arrow), cypermethrin group, 45th day, bar = 50 µm, HE.

Şekil 2.

a. Beyin, kontrol grubu, bar = 50 µm, HE.

b. Fokal gliozis (ok) cypermethrin grubu, 45. gün, bar = 50 µm, HE.

Gözde Yücel - İbrahim Ayhan Özkul 404

Figure 3.

a. Liver, control group, bar = 50 µm, HE.

b. Hyperemia in central venous (arrow) and degeneration of hepatocytes (arrow head), cyphenothrin group, 15th day, bar = 50 µm, HE.

Şekil 3.

a. Karaciğer, kontrol grubu, bar = 50 µm, HE.

b. Vena sentraliste hiperemi (ok) ve hepatositlerde dejenerasyon (ok başı) cyphenothrin grubu, 15. gün, bar = 50 µm, HE.

Figure 4.

a. Ovary, control group, bar = 100 µm, HE.

b. Oophoritis with infiltration of lymphocytes and macrophages in the interstitial area (arrow), cyphenothrin group, 30th day, bar = 50

µm, HE.

c. Testicle, control group, bar = 50 µm, HE.

d. Dilated lumens of seminiferous tubules (star), cyphenothrin group 15th day, bar = 50 µm, HE.

Şekil 4.

a. Ovaryum, kontrol grubu, bar = 100 µm, HE.

b. İnterstisyel bölgede lenfosit ve makrofajlardan oluşan ooforitis (ok), cyphenothrin grubu, 30. gün, bar = 50 µm, HE.

c. Testis, kontrol grubu, bar = 50 µm, HE.

d. Dilate seminifer tubul lumeni (yıldız), cyphenothrin grubu 15. gün, bar = 50 µm, HE.

Ankara Üniv Vet Fak Derg, 63, 2016 405

Table 1. The number of fish in the cypermethrin group showing microscopic changes on each day (n = 7).

Tablo 1. Cypermethrin grubunda günlere göre mikroskobik bulgu görülen balık sayıları (n = 7).

Groups (n = 7)

Lesions 15th day 30 th day 45 th day 60 th day 75 th day

Hyperemia in gill 7 7 7 5 5

Telangiectasia in gill 3 0 0 0 0

Epithelial lifting in gill 4 7 5 7 7

Hemorrhage in gill 0 3 0 0 0

Branchitis in gill 7 3 3 0 0

Passive hyperemia in liver 1 1 0 0 0

Dejenerasyon in liver 0 0 0 0 1

Hemorrhage in kidney 2 2 1 0 0

Hyperemia in brain 2 2 3 6 6

Gliosis in brain 1 3 2 2 0

Table 2. The number of fish in the cyphenothrin group showing microscopic changes on each day (n = 7).

Tablo 2. Cyphenothrin grubu, günlere göre mikroskobik bulgu görülen balık sayıları (n = 7).

Groups (n = 7)

Lesions 15th day 30 th day 45 th day 60 th day 75 th day

Hyperemia in gill 7 4 7 4 7

Telangiectasia in gill 0 0 0 1 0

Epithelial lifting in gill 7 5 7 3 5

Hemorrhage in gill 0 0 0 0 0

Branchitis in gill 2 4 4 4 2

Passive hyperemia in liver 5 0 0 0 1

Dejenerasyon in liver 2 0 0 0 2

Hemorrhage in kidney 2 0 0 0 1

Hyperemia in brain 7 2 3 2 5

Gliosis in brain 1 0 1 1 1

17β-Estradiol (E2): Edema in the interstitial area

was also detected in 1 female fish on day 45.

Degeneration was observed in the seminiferous tubules

of 1 fish in this group on day 15 and in 1 of the 2 male

fish on day 30.

Discussion and Conclusion

Pesticides are often used in agriculture to kill pests,

either to reduce their impact or to bring them under

control. It is known that synthetic pyrethroids are safer

for humans and other mammals than other pesticides.

However, even when these compounds are used in small

amounts, they can enter aquatic systems and be absorbed

by fish, particularly freshwater fish that are consumed by

humans. Therefore, in this study, we investigated the

effects of these pesticides on common carp, as this sturdy

fish is heavily consumed by people.

It has been widely reported that the effects of

cyphenothrin and cypermethrin are mostly seen in the

gills of fish (4, 3, 11, 14, 17). In the present study, we

found that both these substances may lead to shortness of

breath in common carp, although the levels of hyperemia

and telangiectasia in the gills were not consistent over the

course of the study, with no linear increase or decrease

with increasing exposure period. However, epithelial

lifting as a result of swelling was detected in most fish in

both treatment groups. Similar to the argument present

by Lakani et al. (1) our study proves that epithelial lifting

in the secondary lamellae functions as a defense

mechanism by diminishing the interaction between the

surface of the lamellae and the surroundings by

increasing the diffusion barrier. Branchitis was observed

in both treatment groups, but the incidence of this

decreased through the course of the study in the

cypermethrin group, possibly indicating that carp can

tolerate this chemical over time.

Although cypermethrin and cyphenothrin were

applied at sublethal doses, lesions were observed in the

gills, as usually occurs with acute doses. This can be

explained by the argument presented by Velisek et al.

Gözde Yücel - İbrahim Ayhan Özkul 406

(13) that synthetic pyrethroids are strongly absorbed by

the gills, even when present in only small amounts, due

to their lipophilic nature.

We also observed passive hyperemia and

degeneration in the liver, gliosis in the brain, and

hemorrhage in the kidneys. However, these are not

considered to be associated with exposure to pyrethroids

as they were seen in few fish that had lesions.

Unfortunately, we were unable to determine the

impact of cypermethrin and cyphenothrin on the

endocrine system of carp by examining the gonads

because the juveniles used in this study were not fully

developed and their sex could not be determined

accurately. However, the observation of degeneration and

edema in some gonads indicates that additional research

should be performed to evaluate the effect of these

substances over a longer period of time on fish with fully

developed gonads.

Acknowledgements

The authors thank Insecticide Testing Laboratory,

Hacettepe University, Ankara, Turkey for a generous gift

of 93.1% cyphenothrin and 93% cypermethrin used in

the experiments.

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Geliş tarihi: 03.11.2015 / Kabul tarihi: 01.12.2015

Adress for correspondence:

Dr. Gözde YÜCEL

Ankara University, Faculty of Veterinary Medicine

Department of Pathology,

06110 Dışkapı/Ankara, Turkey.

e-mail: gozdeyucel@gmail.com